The vertebrate neuromuscular junction is one of the most accessible and quantitative model synapses for studying cholinergic functions. We are continuing our studies on the family of acetylcholinesterase (AChE) molecules because they are identified synaptic components whose synthesis, assembly, and regulation can be analyzed directly and quantitatively, and, in broader terms, because findings concerning their biogenesis, transport, and localization at the molecular level will be generally applicable towards understanding the regulation of synaptic components in all electrically-excitable cells. We have now developed techniques that allow us to quantitate AChE mRNA, enzyme activity, and a second messenger system involved in its regulation at the level of single neuromuscular synapses. The new approaches will enable us to test models of AChE gene regulation at the molecular and cellular level using adult skeletal muscle fibers and examine quantitatively the regulation of a specific synaptic component at single vertebrate synapses. The specific aims of this proposal are: 1) To study the expression of different AChE transcripts in fast and slow skeletal muscle fibers types and test the hypothesis that the different transcripts arise from separate fiber type-specific promoters regulated by nerve impulse frequency. 2) Examine quantitatively the neural control of AChE mRNA and protein expression in innervated and non-innervated regions of single muscle fibers to determine whether denervation affects AChE activity by increasing extrajunctional enzyme to levels found at the neuromuscular synapse. 3) To test the hypothesis and a model that expression of AChE mRNA at the neuromuscular junction is intermittent rather than constitutive and regulated by a mechanism linked to muscle activity. 4) To study the mechanisms responsible for targeting and localization of AChE at the neuromuscular synapse using a chimeric viral coat protein-AChE fusion protein expressed in muscle cells. 5) To continue our studies on the structure of the AChE gene. 6) To study the relative contributions of transcription and message stabilization to the maintenance of steady state AChE mRNA levels under different activity states of the muscle. And 7) To study the activation of second messenger systems responsible for regulating AChE expression and to localize these systems in relation to acetylcholine receptor clusters in tissue-cultured muscle cells and in vivo. Together these approaches will permit a molecular dissection of the events leading from synaptic activation to plastic changes in the expression of genes encoding synaptic components at an identified vertebrate synapse.